Passivated and stabilized porous mineral oxide supports and methods for the preparation and use of same

ABSTRACT

This invention relates generally to modified porous solid supports and processes for the preparation and use of same. In particular, passivated porous mineral oxide supports are disclosed that are stabilized against leaching under harsh environmental conditions by the application of a thin, protective polymeric coating atop their porous surfaces and that are characterized by a reversible high sorptive capacity substantially unaccompanied by non-specific adsorption of or interaction with biomolecules. Passivation is achieved by the use of a passivation mixture comprising a main monomer, a passivating monomer that is different from the main monomer, and a crosslinking agent, which mixture upon polymerization results in the substantial elimination of the undesirable non-specific interaction between biomolecules and the protective polymeric coating on the surfaces of the mineral oxide matrix.

This is a division of application Ser. No. 07/956,404, filed Oct. 5,1992, now U.S. Pat. No. 5,268,097, which, in turn, is acontinuation-in-part of Ser. No. 07/901,326, filed Jun. 19, 1992, nowabandoned.

TECHNICAL FIELD

This invention relates generally to modified porous solid supports andprocesses for the preparation and use of same. In particular, passivatedporous supports are disclosed which are characterized by a reversiblehigh sorptive capacity substantially unaccompanied by non-specificadsorption of or interaction with biomolecules such as proteins,polysaccharides or oligo- or polynucleotides. Moreover, the passevatedporous supports of the present invention exhibit other characteristicshighly desirable in chromatographic applications, such as high porosity,physical rigidity, high charge density, and chemical stability under avariety of extreme conditions. The passivated porous supports of thepresent invention may also be used advantageously in a high flow, highefficiency mass transfer chromatographic technique which may be carriedout in a fluidized-bed, packed-bed, or other mode of operation.

BACKGROUND OF THE INVENTION GENERAL CONSIDERATIONS

Polyfunctional macromolecules, such as proteins, can be purified by avariety of techniques. One of these techniques is known as ion-exchangechromatography. In ion-exchange chromatography, proteins are separatedon the basis of their net charge. For instance, if a protein has a netpositive charge at pH 7 it will bind to a negatively chargedion-exchange resin packed in a chromatography column. The protein can bereleased, for example, by decreasing the pH or adding cations thatcompete for binding to the column with the positively charged groups onthe protein. Thus, proteins that have a low density of net positivecharge, and thus a lower affinity for the negatively charged groups ofthe column, will tend to emerge first, followed by those having a highercharge density.

Generally, the ion-exchange resins which are used in these proceduresare solids possessing ionizable chemical groups. Two types exist:cation-exchangers, which contain acidic functional groups such assulfate, sulfonate, phosphate or carboxylate, and a second type,anion-exchangers, which contain functional groups such as tertiary andquaternary amines. These ionizable functional groups may be inherentlypresent in the resin or they may be the result of the chemicalmodification of the organic or mineral solid support.

Organic ionic-exchangers which are made from polysaccharide derivatives,e.g., derivatives of agarose, dextran and cellulose, etc., have beenused for both laboratory and industrial scale ion-exchangechromatography. However, these ion-exchangers have many disadvantages.First, polysaccharide-derived ion-exchangers are not very mechanicallystable and are not resistant to strong acids. This instability limitsthe length of the column and, also, limits the flow rate through thecolumn.

Second, such ion-exchangers have limited sorption capacity due to thelimited number of ionic or ionizable groups that can be attached to thepolysaccharide.

Third, these polysaccharidic derivatives are poor adsorbents for use inrapid fluidized-bed separations because of the low density of thematerial. In a fluidized bed it is desirable to pass the fluid withoutsimultaneously washing out the particles. Therefore, it is generallydesirable to have as great a density difference as possible between thesolid support particles (e.g., silica) and the fluidizing medium.

The intrinsic high density of inorganic sorbents based on passivatedmineral substrates facilitates packing and rapid decantation intochromatographic columns. Dense packing prevents formation of emptyspaces and channeling when using packed beds. On the other hand,fluidization of dense particles in aqueous suspension is possible athigh flow rates that, in turn, are very desirable when dealing withlarge scale applications. Operation of fluidized beds at highsuperficial flow velocities is generally not possible with low-densityorganic or polymeric sorbents, which can be elutriated from fluidizedbeds at relatively low liquid flow rates.

On the other hand, synthetic polymers are mechanically more stable thaninorganic supports, and the former are more resistant to strong acidicconditions. However, they suffer disadvantages as well, such as limitedcapacity, limited solute diffusivity and thus, limited productivity.These synthetic polymers also suffer to some extent from the problem ofnon-specific adsorption of biomolecules, such as proteins. Untreatedmineral supports such as silica are also inadequate in manychromatographic protein separation applications because of suchnon-specific adsorption.

Non-specific adsorption is caused by the interaction of a protein withthe surface of the support--be it organic or inorganic in nature. Forexample, silica is an acidic compound, and the negatively chargedsilanol groups present at the solid/liquid interface tend to create aseparate ion-exchange interaction between the surface of silica and theprotein. Non-specific adsorption is also caused by hydrogen bonding thattakes place between, e.g., amino groups present in the amino acidresidues of proteins and these same silanols present at the silicasurface. Such non-specific interactions create separation problemsduring chromatography--e.g., poor protein recovery and/or inadequateresolution. An important objective in the design of a chromatographicseparation is generally to ensure a "single-mode" process of adsorption.However, the ion-exchange behavior associated with surface silanols cancreate a "mixed mode" adsorption system which makes the separation ofbiomolecules much more difficult. Although the sorption capacitygenerated by ionic silanol groups is low, the intensity of theinteraction between the silanol groups and proteins can be high. Theseinteractions therefore have the potential to cause denaturation ofcertain proteins.

Finally, both polysaccharides and most hydroxyl-containing syntheticsorbents are sensitive to the cleaning solutions used in industrialsettings, which often include strong oxidizing agents such ashypochlorite or peracetic acid and which may be characterized byextremes of pH.

Thus, there is an important need for the development of improvedpassivation methods for the treatment of the surfaces of both polymericand inorganic chromatographic supports in contact withprotein-containing solutions, which method is capable of preventing orminimizing such non-specific interactions between proteins and thechromatographic support in order to improve the efficiency ofchromatographic processes.

PREVIOUS EFFORTS AT COATING SOLID SUPPORTS

Several previous investigators have sought to passivate variousmicroporous media including membranes and particulate chromatographicsupports by applying thin surface coatings to inorganic ororganic/polymeric substrates. For example, Steuck, in U.S. Pat. No.4,618,533, discloses a porous polymeric membrane substrate fashionedfrom a thermoplastic organic polymer upon which a permanent coating isgrafted and/or deposited on the entire membrane surface. Thepolymerization and crosslinking of the polymerizable monomer upon andwithin the porous membrane substrate is performed in such a way that athin coating is deposited upon the entire surface of the porousmembrane, including the inner pore walls. Significantly, the porousconfigurations of the coated, composite membrane structures claimed bySteuck are essentially identical to those of the corresponding uncoatedporous membrane substrates, implying that the polymer of Steuck isapplied as a thin surface layer or coating that does not interfere withthe porosity or flow properties of his composite membranes. Moreover,Steuck does not disclose the concept of a "passivating layer" or the useof monomers capable of functioning as "passivating" monomers within themeaning of the present invention as discussed in more detail below.

Varady et al., in U.S. Pat. No. 5,030,352, disclose pellicular supportmaterials useful as chromatography media which are obtained by applyingvarious thin hydrophilic coatings to the surfaces of hydrophobic polymersubstrates (e.g., polystyrene). Varady's surface coatings are applied byfirst exposing the surfaces of the hydrophobic substrate to a solutionof a solute characterized by interspersed hydrophilic and hydrophobicdomains; contact between surface and solute takes place under conditionsthat promote hydrophobic-hydrophobic interaction between solute andsubstrate, with the result that solute molecules are adsorbed onto thesurface of the substrate as a thin coating that is ultimatelycross-linked in place. Varady's coating materials may further comprisereactive groups capable of being derivatized to produce variousmaterials useful in ion-exchange, affinity, and other types ofchromatographic and adsorptive separations.

Significantly, however, the hydrophilic, functional coating of Varady'sinvention is limited to a thin adherent film on the surface of thehydrophobic support. The morphology of this coating layer is a directand unavoidable consequence of the stated method of itsdeposition--i.e., by the crosslinking of adjacent solute moleculesadsorbed onto the surface of the hydrophobic substrate.

While Varady's coating method is at least partially effective inreducing the non-specific binding of proteins to the substrate, thesorption capacity of the chromatographic materials so produced isnecessarily limited and inferior to those of the media produced by theprocess of the present invention. As discussed in considerably moredetail below, the method of the present invention causes the formationof a crosslinked and functional gel that extends out into andsubstantially fills the pores of the support. As a consequence, thestatic and dynamic sorption capacities of the chromatographic media arenot limited by the porous surface area of the substrate, as is the casewith the pellicular materialq of Varady's invention.

With regard to previous techniques for the passivation of inorganic ormineral supports by surface coating treatments, U.S. Pat. No. 4,415,631to Schutijser discloses a resin consisting of inorganic silanizedparticles onto which is bonded a cross-linked polymer comprised ofcopolymerized vinyl monomers and which contains amide groups. Theinvention specifies that the inorganic porous support, including silica,must be silanized prior to coating. The silanization treatment providesthe inorganic porous support with reactive groups so that the copolymercan be covalently bonded to the silica surface.

Nakishima et al., in U.S. Pat. No. 4,352,884, also discloses the use ofsilica as a porous substrate. The silica is coated with a polymer madeup of acrylate or methacrylate monomer and a copolymerizable unsaturatedcarboxylic acid or a copolymerizable unsaturated amine. Nakashima et al.use an already preformed polymer to coat the support. Furthermore,Nakashima et al., in a separate and distinct step, utilize acrosslinking agent in a subsequent curing process.

The above-mentioned inventions are not completely successful, partlybecause of the unstable chemical linkage between the silica and thecoating. The products of these inventions have the further disadvantagesof not only failing to totally suppress the initial non-specificadsorption but also of introducing additional modes of nonspecificadsorption.

Tayot et al., in U.S. Pat. No. 4,673,734, disclose a porous mineralsupport that is impregnated with an aminated polysaccharide polymer thatis said to cover the internal surface area of the support. However,since polysaccharides usually have very large molecular weights andtheir solutions are quite viscous, this process is not highly effective.Coverage of the entire internal surface of the silica substrate isproblematic due to incomplete and uneven filling of the pores of thesilica substrate by the large polysaccharide molecules.

The steric problems of Tayot's process result from the large size of thepolysaccharides employed, the chains of which cannot penetratecompletely within the pores of the support. This incomplete penetrationresults in the creation of a "soft" layer of polysaccharide on thesurface of the pore that subsequently causes problems duringchromatographic separation. Polysaccharides such as dextran can alsospontaneously hydrolyze at low pH, rendering them incompatible withcertain cleaning operations that require the column or bed ofchromatographic media to be washed with acid, alkaline, or oxidizingagents.

Despite these and other problems associated with the use of inorganicchromatographic suports, the use of mineral compounds such as silica assupports for chromatographic adsorbents is still attractive, because asexplained above, chromatographic separations can be performed with suchmaterials at very high flow rates--for example, in very large-scalepacked columns or in fluidized beds for industrial operations. What isneeded are chromatographic supports characterized by high static anddynamic sorption capacity which exhibit improved chemical stability atalkaline and basic conditions and reduced tendencies to causenon-specific protein adsorption. It is an object of the presentinvention to provide such supports.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a passivated porous supportcomprising a porous solid matrix having interior and exterior surfacesand innate (i.e., inherently present) groups that render the matrixsusceptible to undesirable non-specific interaction with biologicalmolecules, and a polymer network derived from a passivation mixturecomprising effective amounts of a main monomer, a passivating monomerdifferent from the main monomer, and a crosslinking agent, the mixturehaving been allowed to come into intimate contact with the surfaces ofthe matrix for a sufficient period of time such that on polymerizationof the mixture the innate groups of the matrix become deactivated,resulting in the minimization or substantial elimination of theabove-mentioned undesirable non-specific interactions.

The passivated porous supports of the present invention are furthercharacterized by reversible high sorptive capacity for biologicalmolecules including proteins. Furthermore, the passivated poroussupports of the present invention enjoy exceptional chemical stabilityon exposure to strongly acidic or alkaline media and/or strong oxidizingsolutions such as those that are frequently utilized during cleaning ofindustrial manufacturing equipment.

The primary objective of the present invention concerns the passivationof porous solid matrices that possess innate undesirable groupsthatrender the matrix susceptible to non-specific interactions (e.g.,adsorption) with biological molecules, in particular, proteinaceoussubstances.

A wide variety of non-passivated porous solid matrices are amenable topassivation by the general method of the present invention. These porousmatrices include, but are not limited to, (i) mineral oxide supports,(ii) "stabilized" mineral oxide supports rendered chemically resistantto leaching by the application of thin protective coatings ofhydrophobic polymers to their surfaces, and (iii) porous 0 matricescomprised solely of organic/polymeric materials, in particularhydrophobic polymers.

For example, mineral oxide supports, such as silica, alumina, and thelike, may be transformed into passivated supports that exhibit desirablecharacteristics, such as high sorptive capacity, high density and goodresolving (chromatographic) properties, unaccompanied by undesirablenon-specific interactions that would otherwise be due largely to innatehydroxyl groups present on the surfaces of mineral oxides (e.g.,silanols in the case of silica supports). It should be noted thattransition metal oxides, such as zirconium, titanium, chromium and ironoxides are considered in the present invention to be within the scope ofthe term "mineral oxide" supports.

In the case of such mineral oxide supports, the non-specificinteractions include either electrostatic interactions, hydrogenbonding, or both. Hence, the passivating monomer (alternativelydescribed herein as the "neutralizing" monomer) is chosen to dampen,"neutralize" or "deactivate" such non-specific binding interactions;that is, one selects a passivating monomer that is capable ofinteracting with the innate groups of mineral oxide substrates eitherelectrostatically or via hydrogen-bonding or both.

Moreover, in particular embodiments of the present invention, thepassivating monomer can also act as the main monomer (i.e., saidpassivating or neutralizing monomer is chemically identical to the mainmonomer), but such situations are limited to those in which theneutralizing monomer is an acrylamide-based monomer that possesses atleast one polar substituent, preferably an ionizable (e.g., tertiaryamino, carboxylic acid, sulfonic acid, etc.) or ionic (e.g., ammonium,phosphate, etc.) substituent. In particular, acrylate-based monomerscannot serve both as the passivating (neutralizing) monomer and as themain monomer--in part, because the acrylate-based monomers are lessstable than the acrylamide-based monomers, particularly under stronglyacidic or alkaline conditions.

Without wishing to be by theory, it is believed that the utilization ofa passivating or neutralizing monomer, in combination with the mainmonomer and crosslinking agent, allows for the formation of athree-dimensional polymer network comprising a thin passivation regionor layer that is substantially adjacent to the matrix surface, whichpolymer network extends into and throughout the porous volume of thesubstrate matrix and which passivation layer is made up primarily ofunits of the passivating or neutralizing monomer engaged in interactionswith the innate groups of the substrate matrix. This thin passivationregion or layer is additionally held in close proximity to the matrixsurface by a lattice of main monomer units which extends from thepassivation layer to the exposed exterior surfaces of the resulting"passivated" porous support. In addition, the crosslinking agent acts totether the respective polymeric (or copolymeric) chains to one another,thereby creating a stable three-dimensional polymer (i.e., "gel")network that is surprisingly effective in minimizing or eliminatingundesirable non-specific binding interactions between biologicalmolecules and the non-passivated porous solid matrix.

Thus, it is also an object of the present invention to provide apassivated porous support comprising a porous solid matrix havinginterior and exterior surfaces and innate groups that render the matrixsusceptible to undesirable, non-specific interaction with biologicalmolecules, and a polymer network derived from a passivation mixturecomprising effective amounts of an acrylamide or methacrylamide monomerfurther substituted with at least one polar ionic or ionizablesubstituent, which monomer is capable of functioning both as a mainmonomer and as a passivating or neutralizing monomer, and a crosslinkingagent, the mixture having been allowed to come into intimate contactwith the surfaces of the matrix for a sufficient period of time suchthat on polymerization of the mixture, the innate groups of the matrixbecome deactivated, resulting in the substantial elimination of theabove-mentioned undesirable non-specific interaction. Where porousmatrices comprised of hydrophobic polymer substrates (as opposed tomineral oxide matrices) are concerned, it is a further object of thepresent invention to reduce the non-specific binding associated withexposure of such hydrophobic polymer surfaces to proteinaceoussolutions. In particular, porous synthetic polymeric solid matricescomprised of such materials as polystyrene, polysulfone,polyethersulfone, polyolefins (e.g., polyethylene and polypropylene),polyacrylate, polyvinyl acetate (and partially hydrolyzed versionsthereof), and the like, exhibit non-specific binding associated withhydrophobic-hydrophobic (among other types, e.g., hydrogen-bonding)interactions. Unlike the case of the mineral oxide matrix, in which theneutralizing monomer component of the passivating mixture is selected todeactivate polar groups like silanols, hydrophobic synthetic polymermatrices are passivated by the incorporation of passivating("neutralizing") monomers that are capable of associating with andconsequently deactivating innate non-polar hydrophobic groups exposed onthe matrix surface. The passivating monomers of the present inventionadsorb upon (and consequently cover) the hydrophobic groups on thesurface by virtue of their containing long-chain saturated hydrocarbons,olefinic hydrocarbon groups, aromatic groups, or like hydrophobicdomains that interact with and become appreciably bound to theirhydrophobic counterparts on the matrix surface as a consequence of thehydrophobic-hydrophobic interaction between them.

In a further object of the present invention, passivated porous supportsexhibiting exceptional stability in alkaline media are provided. Thesepassivated resins comprise porous solid matrices pre-coated with a thinfilm of a synthetic organic polymer, such as polystyrene or polystyrenesubstituted with nonionic, ionic, or ionizable functional groups. Thesepre-coated matrices exhibit the improved characteristics after beingsubjected to the passivation method disclosed herein.

More particularly, the methods of the present invention can beadvantageously applied to the passivation of chromatographic supportmedia comprised of porous mineral oxide particles (e.g., silica andalumina), the interior and exterior surfaces of which have previouslybeen coated with a thin, protective layer of a coating polymer. Thisprotective polymer coating is applied for the purpose of improving thechemical stability of the underlying mineral oxide material (e.g.,against leaching or other chemical decomposition at alkaline, acidic, orstrongly oxidizing conditions). For example, strongly alkaline aqueousmedia (e.g., 0.5M sodium hydroxide solutions) are commonly used to cleanchromatographic supports, and conventional silica supports can suffersignificant weight loss (of order 50%) associated with leaching of thematerial over repeated cleaning cycles (e.g., 100 cycles).

The leaching of such unprotected mineral oxide supports gives rise to anumber of problems, not the least of which is loss of mechanicalintegrity of the support and a consequent increase in the back pressureexhibited by columns packed with particles of the material. The problemof leaching can be addressed to some extent by using porous matricescharacterized by lower surface areas (e.g., 5-10 m² /g), but this isgenerally undesirable insofar as sorption capacity is often reduced by acorresponding amount.

The approach to substrate stabilization taken in one embodiment of thepresent invention involves coating the alkaline-sensitive porous mineraloxide substrate matrix with a soluble polymer that substantiallyencapsulates the mineral oxide matrix and thereby minimizes or preventscontact between the mineral oxide substrate and potentially destructivechemical cleaning solutions (e.g., caustic). The protective polymercoating is applied in the form of a thin surface layer upon the porewall surfaces in order to avoid significantly decreasing the porousvolume or blocking the mouths of pores. The protective polymer coatinglayer is readily applied, for example, by (i) first dissolving theprotective polymer (e.g., polystyrene) in a suitable organic solvent toform a coating solution, (ii) subsequently impregnating the porousmineral oxide matrix with said solution, and then (iii) finallyevaporating or otherwise removing the organic solvent.

While it has been discovered that this process of depositing protectivepolymer coatings upon the porous surfaces of mineral oxide (andparticularly silica) matrices can significantly stabilize thesematerials by sharply reducing their rates of chemical leaching, theapproach has the important disadvantage of rendering the porous surfacesof the coated and protected matrices hydrophobic and thus prone to causeexcessive non-specific binding of proteins by adsorption. (This isprecisely the same problem noted above in connection with entirelypolymeric porous support matrices.) However, this problem can besuccessfully addressed by the methods of the present invention in thesame way as the non-specific binding of strictly polymeric supportmatrices can be reduced--i.e., by passivation in a process of orientedpolymerization. More particularly, these composite chromatographicsupports (i.e., supports comprised of mineral oxide substrates that havebeen stabilized bythe application of thin protective polymer coatings)can be passivated against excessive non-specific binding byincorporating passivating ("neutralizing") monomers capable ofassociating with and consequently deactivating innate non-polarhydrophobic groups exposed on the matrix surface. The passivatingmonomers useful in this embodiment of the present invention adsorb upon(and consequently cover) the hydrophobic groups on the surface by virtueof their containing long-chain saturated hydrocarbons, olefinichydrocarbon groups, aromatic groups, or like hydrophobic domains thatinteract with and become appreciably bound to their hydrophobiccounterparts on the matrix surface as a consequence of thehydrophobic-hydrophobic interaction existing between them. Typically,the present invention utilizes. base matrices having the followingcharacteristics: an initial average particle size ranging from about 5to about 1000 microns; an initial porous volume ranging from about 0.2to about 2 cm³ /gram; an initial surface area ranging from about 1 toabout 800 m² /gram; and an initial pore size ranging from about 50 toabout 6000 angstroms. Preferably, the base matrix is characterized by:an initial average particle size ranging from about 10 to about 300microns, although passivated supports having narrow particle sizeranges, such as about 15-20, about 15-25, about 30-45, about 50-60,about 80-100, and about 100-300 microns, are most preferred. Preferredranges for other characteristics include an initial porous volumeranging from about 0.8 to about 1.2 cm³ /gram; an initial surface arearanging from about 10 to about 400 m² /gram; and an initial pore sizeranging from about 1000 to about 3000 angstroms. The density of theporous solid matrix obviously varies with its chemical nature, beinghigher for mineral oxide (e.g., silica) substrates and lower forpolymeric ones (e.g., polystyrene).

The size exclusion limit varies somewhat from one type of passivatedporous support to another, but generally falls in the range of about 500to about 2,000,000 daltons, preferably, 50,000 to about 500,000. Thesorptive capacity can also be manipulated, depending on the amount ofmain monomer incorporated in the polymer network, and ranges betweenabout 1 milligram to about 300 milligrams of solute or biologicalmolecule per unit volume (ml) of passivated support bed--preferably atleast about 50 mg/ml, and most preferably about 100 mg/ml.

Yet another object of the present invention relates to the passivationof non-passivated porous solid matrices while maximizing the openness(e.g., gel porosity and pore size) of the resulting passivated poroussupport. Such open gel morphologies have the advantage of permittinghigh sorption capacities to be achieved without affording excessiveresistance to the transport of solutes such as proteins through the gel.Hence, in particular embodiments of the present invention, thepolymerization of the passivation mixture is effected in the presence ofan effective amount of a pore inducer.

A number of additives are suitable as pore inducers, including, but notlimited to, polyethylene glycol, polyoxyethylene, polysaccharide, andthe like. Also, the polymerization of the passivation mixture can beeffected in the presence of an effective pore-inducing amount of a polarsolvent. For example, the polymerization can be carried out in alcohol,a cyclic ether, a ketone, a tertiary amide, a dialkyl sulfoxide, ormixtures thereof. Preferably, such polar solvents include, but are notlimited to, methanol, ethanol, propanol, tetrahydrofuran,dimethylsulfoxide, dimethylformamide, acetone, dioxane, or mixturesthereof.

According to the present invention, polymerization is effected in thepresence of an effective amount of a polymerization initiator, forexample, thermal initiators such as ammonium persulfate/tertiary amine,nitriles or transition metals. Other examples include2,2'-azobis(2-amidinopropane) hydrochloride, potassiumpersulfate/dimethylaminopropionitrile, 2,2'-azobis(isobutyronitrile),4,4'-azobis(4-cyanovaleric acid), or benzoylperoxide. Photochemicalinitiators may also be used, such as isopropylthioxantone,2-(2'-hydroxy-5'-methylphenyl) benzoltriazole,2,2'-dihydroxy-4-methoxybenzophenone, riboflavin, and the like.Polymerization begins, as is known in the art, e.g., with agitation,exposure to heat, or exposure to a sufficient amount of radiant energy.

It is the object of the present invention to provide further passivatedporous supports in which the main monomer of the polymer networkcomprises a vinyl monomer having at least one polar substituent. Suchsubstituent may further be ionic, non-ionic, ionizable, or in the caseof a vinyl monomer having more than one polar substituent, suchsubstituents may be a combination of such substituents. It is preferredin affinity chromatography that the main monomer on polymerization, aspart of the polymer network, have an affinity for a preselectedbiological molecule. However, the further modification of the polymernetwork to incorporate specific ligands capable of binding to biologicalmolecules of interest is not precluded.

It should be apparent to one of ordinary skill in the art that thesubstituent(s) on the passivating or neutralizing monomer responsiblefor the "deactivation" (i.e., the reduction in the capacity of theinnate groups of the non-passivated porous solid matrix to interact in anon-specific manner with biological molecules) should be tailored to thenature of the non-specific interaction to which the non-passivatedporous solid matrix is susceptible. In essence, neutralizing monomersare provided which can interact with the innate groups of the matrixsurfaces in the same manner as the non-specific interaction (e.g.,electrostatically, via hydrogen bonding or both in the case of mineraloxide matrices--or via hydrophobic-hydrophobic interaction in the caseof synthetic polymeric matrices). Hence, substituents can be polar,cationic, anionic or hydrophobic depending on the particular applicationat hand. For example, , suitable neutralizing monomers for porousmineral oxide matrices comprise a vinyl monomer having at least onepolar ionic or ionizable substituent. In one embodiment of the presentinvention, the substituent has the capacity to bear a positive charge.In particular, such neutralizing monomers are selected to providenear-surface passivating regions and polymer networks that are effectivein deactivating-polar groups on the surfaces of non-passivated matrices(e.g., in deactivating hydroxyl groups on the surfaces of porous mineraloxide matrices).

As a non-limiting example, neutralizing monomers useful in thepassivation of porous mineral oxide matrices may be selected fromdiethylaminoethyl methacrylamide, diethylaminoethyl acrylamide,methacrylamido propyltrimethyl ammonium halide, triethylaminoethylacrylamide, trimethylaminoethyl methacrylate, polyethyleneglycoldimethacrylate, dimethylaminoethyl methacrylate, polyethyleneglycoldivinyl ether, or polyethyleneglycol diacrylate. Of these, the firstfour can function within the same composition both as a main monomer anda neutralizing monomer, as discussed above.

Likewise, suitable passivating monomers for use in the passivation ofhydrophobic polymer surfaces--whether said polymer is present as aprotective surface coating on a mineral oxide matrix or as the bulk,structural material in the case of a porous polymeric chromatographicsupport matrix--will typically comprise vinyl monomers having at leastone substantially non-polar or hydrophobic substituent. In oneembodiment of the present invention, this substituent comprises ahydrocarbon-rich functional group or moiety that imparts hydrophobicityto a portion of the passivating monomer.

In general, the hydrophobic character will result from the presence inthe passivating monomer of a saturated (e.g., aliphatic) or unsaturated(e.g., aromatic) hydrocarbon substituent, and may further be describedas straight-chain, branched, cyclic, or heterocyclic. Long-chain alkylfunctional groups are particularly useful as substituents in this classof passivating monomers, which further contain one or more vinylic,acrylic, acrylamide, or allylic monomers. These passivating monomers aretypically employed at concentrations in the reaction mixture of fromabout 0.1 to 1.0%.

Crosslinking agents useful in the present invention comprise vinylmonomers having at least one other polymerizable group, such as doublebond, a triple bond, an allylic group, an epoxide, an azetidine, or astrained carbocyclic ring. Preferred crosslinking agents having twodouble bonds include, but are not limited to,N,N'-methylenebis-(acrylamide), N,N'-methylenebis(methacrylamide),diallyl tartradiamide, allyl methacrylate, diallyl amine, diallyl ether,diallyl carbonate, divinyl ether, 1,4-butanedioldivinylether,polyethyleneglycol divinyl ether, and 1,3-diallyloxy-2-propanol.

It is a further object of the present invention to provide a method ofpassivating a porous solid matrix having interior and exterior surfacesand innate groups that render the matrix susceptible to undesirablenon-specific interaction with biological molecules, comprising: (a)contacting the surfaces of the matrix with a passivation mixturecomprising effective amounts of a main monomer, a neutralizing monomerdifferent from the main monomer, and a crosslinking agent; and (b)effecting the polymerization of the mixture to form a three-dimensionalpolymer network within the pores of the matrix, such that the innategroups of the matrix become deactivated, resulting in the substantialelimination of undesirable non-specific interaction.

In the present method the amount of neutralizing monomer is chosen to besufficient to counteract the innate groups present on the surface of thenon-passivated matrix. Furthermore, the surfaces of the matrix arecontacted (e.g., by dropwise addition) with a solution of thepassivation mixture. Generally, the passivation mixture is prepared asan aqueous solution and, as mentioned above, may in addition containeffective amounts of a pore inducer. In a preferred embodiment of thepresent invention as it is applied to porous mineral oxide matrices, thevolume (in ml) of the passivation mixture solution is adjusted tocorrespond approximately to the weight (in grams) of the non-passivatedporous solid matrix.

Yet another object of the present invention is related to a method ofseparating a desired biological molecule from a sample containing samecomprising: (a) loading a column packed with the passivated poroussupport having an affinity for a preselected biological molecule with asample containing the preselected biological molecule; and (b) passingan eluent solution through the loaded column to effect the separation ofthe preselected biological molecule. The sample may be introduced to thecolumn in any number of ways, including as a solution. Chromatographicseparations employing these passivated supports in fluidized-bed modesof operation are also within the scope of the invention.

The methods of the present invention are effective to isolate orseparate a broad range of biological molecules, including peptides,polypeptides, and proteins (such as insulin and human or bovine serumalbumin), growth factors, immunoglobulins (including IgG, IgM, andtherapeutic antibodies), carbohydrates (such as heparin) andpolynucleotides (such as DNA, RNA, or oligonucleotide fragments).

Eluent solutions suitable for use in the present invention are wellknown to those of ordinary skill in the art. For example, a change inionic strength, pH or solvent composition may be effective in "stepwise"elution processes. Alternately, eluent solutions may comprise a saltgradient, a pH gradient or any particular solvent or solvent mixturethat is specifically useful in displacing the preselected biologicalmolecule. Such methods are generally known to those engaged in thepractice of protein chromatography. Still another object of the presentinvention relates to a chromatographic method for the separation ofbiological molecules comprising passing a sample containing a mixture ofbiological molecules through a column packed with the passivated poroussupport disclosed herein.

Moreover, a method of preparing a passivated porous solid support isdisclosed comprising: (a) contacting a porous solid matrix, havinginterior and exterior surfaces and innate groups that render the matrixsusceptible to undesirable non-specific interaction with biologicalmolecules, with a passivation mixture comprising effective amounts of amain monomer, a passivating or neutralizing monomer different from themain monomer, and a crosslinking agent; and (b) effecting thepolymerization of the mixture to form a polymer network within the poresof said porous solid matrix, such that the innate groups of the matrixbecome deactivated, to provide a passivated porous solid support that issubstantially free of undesirable non-specific interactions.

These and other objects of the present invention will become apparent tothose skilled in the art from a reading of the instant disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph which schematically represents the chromatographicseparation of a protein mixture consisting of (1) cytochrome, (2) bovinehemoglobin, (3) ovalbumin, and (4) beta-lactoglobin on a cationicpassivated porous support. The conditions of the experiment were asfollows:

Column Size: 1.0 cm ID×7.8 cm

Initial Buffer: 50 mM Tris-HCl, pH 8.6

Elution gradient: 0-1M NaCl

Flow Rate: 125 ml/h

FIG. 1B is a graph which schematically represents the chromatographicseparation of a protein mixture consisting of (1) ovalbumin, (2)beta-lactoglobulin, (3) cytochrome c and (4) lysozyme on an anionicpassivated porous support. The conditions of the experiment were asfollows:

Column Size: 1.0 cm ID×7.5 cm

Initial Buffer: 50 mM Acetate, pH 6.5

Elution Gradient: 0-2M NaCl

Flow Rate: 150 ml/h

FIG. 2 represents a comparison between the chromatographic separationsof a protein mixture consisting of (1) ovalbumin, (2)beta-lactoglobulin, (3) cytochrome c, and (4) lysozyme using an anionicpassivated porous support and an anionic nonpassivated matrix. Theconditions of the experiment were as follows:

First Buffer: acetate 50 ml, pH 6.5

Second Buffer: acetate 50 ml, pH 6.5 2M NaCl, pH 4.5

Flow Rate: 140 ml/h

FIG. 3A shows a graph of useful relative sorption capacity versus flowrate for various porous supports including the porous supports of thepresent invention passivated with a cationically charged polymer network(i.e., a passivated porous support useful as an anion-exchange resin).

FIG. 3B shows a graph of productivity versus flow rate for the variousporous supports shown in FIG. 3A.

FIG. 4 shows a graph of the absolute sorptive capacity (in mg/ml) as afunction of flowrate of a variety of solid supports, including apassivated porous support of the present invention.

FIG. 5 is a schematic illustration of the putative architecture of thethree-dimensional polymer network formed within and extending from theinternal surfaces of an individual pore in a porous solid matrix uponpolymerization of the passivation mixture of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention requires first the dissolution ofmonomers in water or in an aqueous/organic solution.

A primary component of the passivation mixture of the present inventionis the main monomer. The appropriate amount of main monomer (or othersolute) for use in the present invention is expressed as a percentageequal to the number of grams of main monomer per 100 ml of monomersolution (percent weight/volume). For purposes of the presentdiscussion, the volume of the monomer solution is effectively the volumeof the solution of a passivation mixture containing main monomer,neutralizing monomer, and crosslinking agent. Appropriate concentrationsof the main monomer range from about 5% to about 50% (i.e., 5-50 gramsof main monomer per 100 mL of monomer solution). Preferredconcentrations of the main monomer are from about 7% to about 20%.

For purposes of this application, the main monomer is defined asincluding any monomer known to those skilled in the art which can beutilized for the preparation of an adsorbent useful in a chromatographicseparation (e.g., affinity, ion-exchange, and the like). Such monomersinclude, but are not limited to, non-ionic monomers, ionic monomers,hydrophilic monomers, hydrophobic monomers, and reactive monomers.Reactive monomers are monomers having special functional groups thatenable them to react chemically with other molecules that aresubsequently immobilized irreversibly on the polymer network. Thisprocedure is the basis of affinity chromatography, the chemicallyattached molecule being referred to as the "ligand". The main monomersof the present invention can be aliphatic, aromatic or heterocyclic;however, they must possess a polymerizable double bond; for example, themain monomers can be acrylic, allylic, vinylic or the like.

More specifically, anionic polymers are used to 0 create anionicsorbents (i.e., cation-exchange supports). The functional groups (i.e.,the substituents on the vinyl monomer) are preferably: carboxylic groups(e.g. acrylic acid, N-acryloyl-aminohexanoic acid,N-carboxymethylacrylamide), sulfonate groups (e.g.acrylamidomethyl-propane sulfonic acid), or phosphate groups (e.g.N-phosphoethyl-acrylamide).

Cationic polymers used to create cationic sorbents may contain thefollowing functional groups: substituted amino groups (e.g.,diethylaminoethyl methacrylamide, diethylaminoethyl acrylamide,methacrylamidopropyltrimethylammonium halide, triethylaminoethylacrylamide, trimethylaminoethyl methacrylate, polyethyleneglycoldimethacrylate, dimethylaminoethyl methacrylate, polyethyleneglycoldivinyl ether, or polyethyleneglycol methacrylate), or heterocyclicamines (e.g., 2-vinylpyridine, vinylimidazole, 4-vinylpyridine).Nonionic polymers may be comprised of: acrylamide, hydroxy-containingacrylamide derivatives (e.g. N-tris-hydroxylmethyl-methyl-acrylamide,methylolacrylamide, dimethylacrylamide, 2-hydroxyethylacrylamide,N-acryloyl-morpholine), methacrylamide, hydroxy-containingmethacrylamide derivatives, heterocyclic neutral monomers (e.g.vinylpyrrolidone, N-acryloyl-morpholine), or hydroxy-containingacrylates and methacrylates (e.g. hydroxyethylacrylate or hydroxyethylmethacrylate, hydroxyphenyl methacrylate, 4-vinylphenol, and2-hydroxypropylacrylate).

Hydrophobic monomers useful in creating sorbents for hydrophobicchromatography include octyl-acrylamide or -methacrylamide,phenyl-acrylamide, butyl-acrylamide, benzyl-acrylamide, andtriphenylmethyl-acrylamide.

Activated monomers useful in creating preactivated sorbents (i.e., thosethat can be further derivatized directly with a "ligand") for affinitychromatography include glycidyl-acrylate or -methacrylate, acrolein,acrylamidobutyraldehyde dimethylacetal, acrylic-anhydride, acryloylchloride, N-acryloxysuccinimide, and allyl-chloroformate.

The passivation mixture further comprises an appropriate amount of apassivating or neutralizing monomer capable of neutralizing thenon-specific adsorption properties of innate sites on the surface of theporous solid support. In the case of silica, the acidic character ofinnate silanol groups proves problematic during separations, and it isthus desirable to neutralize these silanol groups. The amount ofneutralizing monomer to be used is preferably an amount sufficient tocounteract approximately up to an equivalent number of Si-OH groupspresent at the exterior and interior surfaces of said support. Theamount of neutralizing monomer, again expressed as a percentage(weight/volume), should be about 0.5% to about 6% (w/v), preferablyabout 1.5 to about 3% (i.e., about 1.5-3 grams of neutralizing monomerper 100 ml of monomer solution).

Suitable neutralizing monomers for use in the present invention may bemonomers bearing a positive charge at a neutral pH; examples includemonomers containing a cationic amine group, such as substituted aminesor pyridine and the like. The cationic neutralizing monomers must haveat least one double bond, such as vinyl, acrylic, or allylic monomers.

To counteract the acidic character of silica and its tendency to formhydrogen bonds, cationic monomers or monomers which are able to engagein hydrogen bonding (dipolar interactions) are also useful asneutralizing monomers in a particular embodiment of the presentinvention.

Preferred neutralizing cationic monomers of the present inventioninclude, but are not limited to, diethylaminoethyl acrylamide,diethylaminoethyl methacrylamide, diethylaminoethyl methacrylate,methacrylamide propyltrimethyl ammonium halide, triethylaminoethylacrylamide, triethylaminoethyl methacrylate and copolymers thereof.

Polyoxyethylene-containing monomers can also be used. This latter groupcan interact with polar groups (via hydrogen bonding). Preferredneutralizing monomers able to induce hydrogen bonding arepolyoxyethylene monomers like poly(ethylene glycol)_(n)-dimethylacrylate, where "n" is between about 50 and about 1000.

Preferred neutralizing hydrophobic monomers include, but are not limitedto, N-alkylacrylamide in which the alkyl groups are branched,N-alkylacrylamide methylene chains having up to about 20 carbon atoms inthe alkyl moiety, and N-arylacrylamide derivatives, likeN-benzylacrylamide, N,N-(1,1-dimethyl-2-phenyl)ethyl-acrylamide,N-triphenyl methylacrylamide, or N,N-dibenzyl acrylamide. Specificrepresentative passivating monomers useful in treating polymeric orpolymer-coated matrices include, but are not limited to,N-tert-octylacrylamide (TOA), N-(1-methylundecyl)acrylamide (MUA),N-(1,1,3,5-tetramethyl)-octylacrylamide (TMOA),Triton-X-100-methacrylate (TWMA) i.e., a methacrylate of an octylphenoxypolyethoxylethanol available commercially from Union Carbide Corp. underthe tradename Triton-X-100, and polyethyleneglycol-dimethacrylate(PEG-DMA). Hydrophobic adsorption sites present on the internal surfacesof some organic (i.e., polymeric) porous matrices like polystyrene--oron protective polymer coatings deposited on porous mineral oxidematrices--are neutralized using hydrophobic passivating monomersincorporating these aromatic and aliphatic hydrophobic moieties orsubstituents.

To the mixture comprising the neutralizing and main monomers, abifunctional crosslinking agent is added. The crosslinking agent allowsthe three-dimensional insoluble polymeric network to form within thepore volume of the porous matrix. In the absence of the crosslinkercalled for in this invention, the polymer formed would be linear andthus soluble. The amount of crosslinking agent should be about 0.1% toabout 10% (w/v). Alternatively, the amount of crosslinking agent can becalculated based on the total weight of main monomer and neutralizingmonomer in use. Preferably, the amount of crosslinking agent is fromabout 3 to about 10 percent by weight of the total weight of main andneutralizing monomers.

The crosslinking agents used in the present invention are acrylic,vinylic or allylic monomers that possess at least two polymerizablefunctional groups. Preferred crosslinking agents have at least twodouble bonds and include, but are not limited to,N,N'-methylene-bis-acrylamide, N,N'-methylene-bismethacrylamide, diallyltartradiamide, allyl methacrylate, diallyl amine, diallyl ether, diallylcarbonate, divinyl carbonate, divinyl ether, 1,4-butanedioldivinylether,and 1,3-diallyloxy-2-propanol.

Thereafter, said mixture is admixed with a porous solid matrix, therebyfilling the pores of the matrix. As regards inorganic support materials,suitable porous mineral oxide matrices used in the present inventioninclude but are not limited to silica, alumina, transition metal oxides(including but not limited to titanium oxide, zirconium oxide, chromiumoxide, and iron oxide) and any other similar ceramic material includingsilicon nitride and aluminum nitride. The preferred mineral moieties ofthe present invention include silica, zirconium oxide, and titaniumoxide. The most preferred mineral moiety is porous silica of a particlesize of about 5 μm to about 1000 μm, having a porous volume of about 0.2to about 2 cm³ /g, a pore size of about 50 to about 6000 Å, and asurface area of about 1 to about 800 m² /gr. At this time, most all ofthe aqueous solution will have been absorbed by the mineral support,leaving a substantially dry, solid porous matrix.

After filling the pores of the porous mineral oxide matrix, (e.g.,silica) with the aqueous solution of monomers (preferably, the volume ofthe solution expressed in mls is approximately equal to the weight ingrams of the silica matrix), the mixture is placed in a non-aqueousdispersing medium. Suitable non-aqueous medium include non-polar organicsolvents known to those skilled in the art. Such non-aqueous medium forsuspending the treated matrix may include but are not limited to mineraland vegetable oils, aromatic solvents, aliphatic low molecular weightsolvents, or chlorinated solvents. The most preferred non-aqueous mediainclude toluene, methylene chloride, and hexane.

Thereafter, a polymerization starter is added to the mixture, now in anon-aqueous medium, in order to initiate polymerization of the monomerswithin the silica pores. The concentration of initiator (expressed aspercent weight per volume of initial monomer solution) is from about0.1% to about 2%, preferably about 0.8% to about 1.2%.

It should be apparent to those of ordinary skill that certain initiatorsare best dissolved in aqueous media while others can be dissolved inorganic media. Hence, depending on the solubility characteristics of aparticular initiator or combination of initiators, the polymerizationinitiator can be added to the initial solution of passivation mixtureprior to addition of that mixture to the porous solid matrix. Inparticular, an initiator combination of ammonium persulfate andtetramethylethylenediamine (TMEDA) can be introduced separately. Onecomponent (the water-soluble persulfate salt) is combined with theaqueous mixture of main monomer, neutralizing monomer, and crosslinkingagent, while the other component (TMEDA) is combined with thenon-aqueous dispersing medium.

It should be noted that the persulfate/TMEDA combination is particularlyuseful because TMEDA displays appreciable solubility in water. Hence, inthe dispersion comprised of the treated support, water and non-aqueoussolvent, the TMEDA is able to penetrate the pores of the treated supportand thereby initiate polymerization, particularly upon heating.

Typical polymerization initiators known to those skilled in the art canbe used in the present invention. For instance, these initiators may becapable of generating free radicals. Suitable polymerization startersinclude both thermal and photoinitiators. Suitable thermal initiatorsinclude but are not limited to ammonium persulfate/tetramethylethylenediamine (TMEDA), 2,2'-azobis-(2-amidino propane)hydrochloride, potassiumpersulfate/dimethylaminopropionitrile, 2,2'-azobis(isobutyronitrile),4,4'-azobis-(4-cyanovaleric acid), and benzoyl-peroxide. Preferredthermal initiators are ammonium persulfate/tetramethyethylenediamine and2,2'-azobis(isobutyronitrile). Photo-initiators include but are notlimited to: isopropylthioxantone,2-(2'-hydroxy-5'-methyl-phenyl)benzotriazole,2,2'-dihydroxy-4-methoxybenzophenone, and riboflavin. It is furthercontemplated that riboflavin be used in the presence of TMEDA. Whenusing the combination of persulfate and tertiary amine, the persulfateis preferably added prior to the addition of the non-aqueous medium,since persulfate is much more soluble in water than in non-aqueousdispersing media.

In another embodiment, the polymerization step can take place in thepresence of a pore inducer. The pore inducers of the present inventionallow polymerization to take place without substantially reducing theporosity of the solid support. Suitable pore inducers,also referred toas porogens, used in the present invention include but are not limitedto polyethylene glycols, polyoxyethylenes, polysaccharides such asdextran, and polar solvents. Polar solvents include those commonly usedin chemical synthesis or polymer chemistry and known to those skilled inthe art. Suitable polar solvents include alcohols, ketones,tetrahydrofuran, dimethylformamide, and dimethysulfoxide. Preferredpolar solvents are ethanol, methanol, dioxane, and dimethysulfoxide.

Porous polymeric matrices amenable to passivation by the methods of thepresent invention include, but are not limited to polystyrene,polysulfone, polyethersulfone, various cellulose esters (e.g., celluloseacetate, cellulose nitrate), polyolefins (e.g., polyethylene andpolypropylene), polyvinylacetate (and partially hydrolyzed versionsthereof), polyacrylates, polyvinylidene fluoride, polyacrylonitrile,polyamides, polyimides, and various blends, mixtures, and copolymersthereof. Procedures for the manufacture of porous particles and otherstructures (e.g., microporous membranes) from such polymers aregenerally known in the art.

Where the polymer surface to be passivated is in the form of a thin,protective coating residing upon the pore walls of mineral oxidesubstrate that is thus stabilized against leaching, the polymer willgenerally consist of a linear, high-molecular-weight polymer capable ofbeing dissolved in a suitable organic solvent. For example, a coatingsolution of linear polystyrene (e.g., with an average molecular weight400 kilodaltons) is conveniently prepared by dissolving the polymer in achlorinated hydrocarbon such as methylene chloride. Typicalconcentrations of polymer in the coating solution range from about 2%(w/v) to about 20% (w/v). The ideal concentration is determined byachieving a balance between effectiveness in preventing or minimizingleaching of the mineral oxide substrate (which argues for higher polymerconcentrations) and the constriction of pores and partial loss of porousvolume (and sorption capacity) that can occur at higher polymerconcentrations. Where protective coatings of polystyrene are depositedon porous silica, a polystyrene concentration of about 10% (w/v) ispreferred. The coating is applied by first impregnating the poroussupport with the solution of protective coating and then removing thesolvent vehicle by evaporation.

Certain modifications to the passivation procedures employed with porousmineral oxide matrices are indicated where the exposed surface of theporous matrix to be passivated is a polymeric one--i.e., in those caseswhere (i) the porous support particle is fashioned entirely of a polymeror (ii) a mineral oxide matrix is protected by a stabilizing polymercoating. In these situations, polymerization of the passivating mixtureby the process described above, entailing the dispersion of the porousparticles (impregnated with aqueous monomer solution) in a non-aqueous(i e., . "oil-phase") dispersing medium, has certain disadvantages. Theproblems stem from the fact that the surfaces of polystyrene-coatedsilica and other polymer-coated mineral oxide matrices are predominantlyhydrophobic and compatible with oil-phase dispersing agents that wouldotherwise be used in the polymerization step. Oil-phase dispersing mediaare prone to penetrating the pores of matrices that present exposedpolymeric surfaces, and the presence of oil inside the pores causesvarious manufacturing problems (e.g., partial solubilization of thecoating polymer, difficulty in effecting removal of the oil from thepores, etc. ).

Accordingly, a modified polymerization procedure is advantageouslyemployed where polymeric surfaces are to be passivated, which procedureentails a so-called "dry polymerization" procedure as opposed to thatdescribed above involving an oil-phase dispersing medium. In particular,the porous matrix impregnated with aqueous passivating mixture (i.e.,monomer solution) undergoes the polymerization reaction while in theform of an apparently "dry" and free-flowing powder, typically agitated(e.g., by stirring or fluidization) in a closed, inert (e.g., nitrogen)atmosphere. The dry polymerization reaction is typically conducted at atemperature from about 60° to 90° C., at a pressure of 1 to 2 bars, andfor a period ranging from about 2 hours to overnight.

Suitably "dry" but monomer-solution-impregnated powders can be preparedby adding the aqueous passivating mixture in a careful, metered fashion(e.g., dropwise) to the porous matrix, so that little or no excessliquid-phase passivating mixture is present. The incorporation oforganic cosolvents (e.g., ethanol, dimethylsulfoxide, and the like) inthe monomer mixture assists the process of wetting the polymeric orpolymer-coated mineral oxide matrix by the predominantly aqueouspassivation mixture. For example, the crosslinking agent is convenientlyadded to the final monomer mixture in the form of an aqueous 10% ethanolsolution.

Because no oil-phase is present as a dispersing medium in thisembodiment of the invention, the initiators (i.e., polymerizationcatalysts) employed in this dry polymerization process are necessarilywater-soluble and are generally thermally activated. A representativethermally-activated polymerization initiator is azo-bis-amidinopropane.

In yet another aspect of the invention, polymeric and polymer-coatedmineral oxide matrices may be treated with hydrophilic polymers such aspolyoxyethylene (POE) and polyvinylpyrrolidone (PVP) prior to effectingthe polymerization and crosslinking of the monomer solution within thepores of the support. Treatment in this manner can be effective inreducing non-specific-binding interactions with proteins even in theabsence of the oriented polymerization of hydrophobically bindingpassivating monomers present in the monomer soluton. Without wishing tobe limited as to theory, it is believed that such high-molecular-weightpassivating polymers are initially adsorbed upon the surfaces of thepolymeric or polymer-coated mineral oxide matrix. Upon polymerization ofthe monomer solution, these polymers become substantially immobilized bythe formation of an interpenetrating polymer network. That is, the POEor PVP polymer becomes entrapped in a "sandwich" type of structurebetween the pore-wall surface and the three-dimensional polymer latticethat occupies most of the porous volume.

In all cases, i.e., whether the porous matrix is comprised of a mineraloxide, a polymer-coated and thus stabilized mineral oxide, or a polymer,the polymerization process of the present invention creates athree-dimensional lattice or cross-linked polymer network that extendsaway from the pore-wall surfaces of the porous solid matrix, . Again,not wishing to be limited by theory, it is believed that this polymernetwork is comprised of a a thin passivating region or layer thatinteracts with the surface of the non-specific adsorption sites of thesolid support (e.g., silanols in the case of silica) covalently linkedwith a three-dimensional structural polymer lattice that substantiallyfills the porous volume. The three-dimensional shape of the polymerlattice is believed to be substantially identical to the shape of thepore which it fills (see FIG. 5), with the passivating layer orientedadjacent to and continuous (i.e., covalently linked) to thethree-dimensional polymer lattice that extends away from the matrixsurface. This configuration prevents "neutralizing" or "deactivating"pieces of the polymer network from eluting from the support duringregular use--for example, when the passivated porous support is exposedto vigorous washing or cleaning conditions, such as high acidic pH, highalkaline pH, high ionic strength, and strong oxidizing conditions. Thiscrosslinked polymer network creates a novel chromatographic sorbentswhich can then be used, for example, in a process for separating andpurifying various biomolecules, including macromolecules.

Indeed, it has been surprisingly discovered that the passivated poroussupports of the present invention manifest chromatographiccharacteristics that are unparalleled under several criteria,particularly in terms of dynamic sorptive capacity as a function of flowrate. In particular, whereas the great majority of porous materialssuffer a marked decrease in useful sorptive capacity as flowratesincrease (e.g., at flowrates of about 50 cm/h or greater), thepassivated porous supports of the present invention show little decreasein useful sorptive capacity from a static condition up to flow ratesapproaching 200 cm/h. Compare, for example, the behavior of prior art"gel"-type materials with the supports of the present invention, asillustrated in the graphs of FIG. 3A, 3B, and 4 (described further inExample 16).

Moreover, the absolute capacities of the passivated porous supports ofthe present invention are considerably greater than even those attainedwith other types of solid supports (e.g., Spherodex™) that exhibit asimilar insensitivity to high flowrates. Thus, as shown in FIG. 4, aplot of the absolute capacity vs. flowrate of various solid supportsunambiguously shows that the passivated solid supports of the presentinvention combine a very high absolute sorption capacity (expressed asmg/ml) with a relative insensitivity to solution flowrates.

It is believed, without wishing to be limited by theory, that a highlyopen, flexible lattice structure comprised primarily of polymeric chainsof repeating main monomer units is formed within the pores of the poroussolid matrix. Very significantly, it is believed that the areas of theporous support available for desirable reversible interaction withbiological molecules are not confined to the regions immediatelyadjacent to the surface of the pore as is the case when thin,substantially two-dimensional coatings are applied to porous surfaces inthe manner of Steuck (U.S. Pat. No. 4,618,533) and Varady et al. (U.S.Pat. No. 5,030,352) as discussed in Section 2.2 above. Rather, it isbelieved that the polymeric network of the present invention extendsoutwardly into the pore volume itself in the manner of athree-dimensional lattice, as opposed to a two-dimensional coatinglimited strictly to the pore wall surface area. A schematic diagram ofsuch a structure, as it is thought to exist, is illustrated in FIG. 5,where a biological molecule of interest (depicted as a spherical object)is also shown interacting with the lattice. Furthermore, the presence ofporogens (pore-inducers) in the passivation mixture is believed topromote creation of this open three-dimensional polymer network.

It is further thought that such an extended polymer network contributesnot only to the unusually high absolute sorptive capacity of thepassivated solid supports of the invention as measured under static(i.e., no flow) conditions, but also allows the present invention tomaintain such high sorptive capacities largely independent of solutionflowrates. It is thought that perhaps the open, flexible nature of thethree-dimensional polymer network allows biological molecules to rapidlypenetrate the polymer lattice and thereby efficiently interact withsorptive groups in the polymer network of the passivated porous supporteven at high solution flowrates. The rapid and efficient mass transferof biomolecules into and through this network avoids the decrease inuseful or dynamic sorption capacity and resolution that are typical ofconventional chromatographic media. With these conventional media,diffusion in the pores of the support and/or materials coated thereuponor within them leads to poor mass transfer rates and limits theefficiency of the chromatographic process.

Thus, a method of performing chromatographic separations characterizedby high sustained sorptive capacity independent of flowrate and rapid,efficient mass transfer is achieved with the passivated porous supportsof the present invention, which supports include an open, flexiblethree-dimensional network or lattice of crosslinked polymer chainsextending within and throughout the pores of the support matrix.

The separation and purification process usually involves at least twosteps. The first step is to charge a packed or fluidized bed columncontaining the passivated porous solid support with a solutioncontaining a mixture of biomolecules, at least one of which it isdesired to separate and recover in at least partially purified form. Thesecond step is to pass an eluent solution through said column to effectthe release of said biomolecules from the column, thereby causing theirseparation.

"Stepwise" elution can be effected, for example, with a change insolvent content, salt content or pH of the eluent solution.Alternatively, gradient elution techniques well known in the art can beemployed. For instance, proteins reversibly bound to cation exchangemedia can generally be eluted by increasing the pH to alkaline values(subject to limits associated with the chemical stability of theprotein), and immunoglobulins bound to protein A or like adsorbents maybe eluted by decreasing the pH to acidic values.

The invention is further defined by reference to the following examplesthat describe in detail the preparation of the passivated porous solidsupport and the methods of using the same. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the purpose and scopeof this invention.

EXAMPLES GENERAL DEFINITIONS

To better understand the procedures described in the following examples,several terms are defined for the benefit of the reader, below.

The passivation level is an estimation of the absence of non-specificadsorption of a strong cationic molecule like lysozyme, whichcharacteristically forms very strong complexes with silanols on thesilica surface.

Porosity factor is the ratio between elution volume (Ve) of a protein(e.g., BSA in our case) and the total volume (Vt) of the packing beddetermined under physiochemical conditions (e.g., high ionic strength)in which no interaction exists between the protein and the poroussupport.

Sorption capacity is the amount of adsorbed protein in "mg" per unitvolume (ml) of passivated porous support bed determined under particularconditions:

for cationic sorbents: 50 m M Tris-HCl, pH 8.6.

for anionic sorbents: 50 mM Acetate, pH 6.5.

Ion exchange capacity is the number of ionizable groups in μeq per unitvolume (ml) of passivated porous support bed determined by titration.

Example 1:

Preparation of a porous cation-exchange resin.

20 grams ("g") of acrylamidomethyl propane sulfonic acid (AMPS) sodiumsalt and 1 g of N,N'-methylene-bis-acrylamide (MBA) are dissolved in 50ml of distilled water. 3 g of diethylaminoethyl methacrylamide, areadded and then the pH of the total solution is adjusted to between 6 to8 to make a final solution volume of the passivation mixture of 100 ml.To this solution of monomers, 500 mg of ammonium persulfate are added atroom temperature.

While shaking, the solution of monomers is added dropwise to 100 g ofporous silica (40 to 100 μm diameters, 1000 to 1500 Å pore diameter, 20to 35 m² /g surface area and 1 cm³ /g porous volume).

After 30 minutes of shaking, 250 ml of paraffin oil is added, theagitated suspension is heated at 60° to 70° C. and then 1 ml ofN,N,N',N'-tetramethylethylene diamine is added.

After a few minutes, the exothermic polymerization reaction occurs. Theresin is then separated by a chlorinated solvent and dried at roomtemperature. Lastly, the resin is washed extensively with dilutehydrochloric acid, dilute sodium hydroxide and 1M sodium chloride.

This cation-exchange resin shows the following characteristics:

A titration curve with an acidic pK due to the presence of sulfonic acidgroups;

No presence of anionic groups which are oriented on the acidic silanolsof the silica surface.

A number of acidic groups of 395 μeq/ml.

A sorption capacity for insulin in 70% ethanol of about 80 mg/ml.

An exclusion limit of about 30 Kd.

EXAMPLE 2

Preparation of an anion-exchange resin.

20 g of methacrylamidopropyl trimethyl ammonium chloride (MAPTAC) and 1g of N,N'-methylene-bis-acrylamide (MBA) are dissolved in 80 ml ofdistilled water and the pH of the solution is adjusted to 7.5.Separately, 1 g of ammonium persulfate is dissolved in 20 ml ofdistilled water. The two solutions were then mixed together at roomtemperature.

While shaking, the monomer solution is added dropwise to 100 g of dryporous silica (40-100 μm bead diameter, 1000-1500 Å porous volume, 20-35m² /g surface area and 1 cm³ /g porous volume).

After shaking for about 30 minutes, 250 ml of paraffin oil is added andthe mixture heated at 60°-70° C. 2 ml of N,N,N',N'-tetramethylenediamineis added to polymerize the monomer solution inside the silica pores.

The same recovery and washing steps are performed as those described inExample 1.

The obtained resin shows the following characteristics:

Ion-exchange capacity: 114 μeq of quaternary ammonium groups per ml ofresin.

No visible presence of acidic (silanol) groups on titration curve.

No non-specific adsorption of cytochrome c at pH below its isoelectricpoint.

Sorption capacity for bovine serum albumin (BSA): 91 mg/ml resin.

Porosity factor for BSA (V_(e) /V_(t)): 0.52.

Example 3

Preparation of a second anion-exchange resin using different amounts ofcross linker.

Three 80 ml solutions each containing two monomers (MAPTAC and MBA) areprepared according to Example 2, using varying amounts of MBA: 0.5 g, 1g and 2 g.

All other operations are identical to Example 2. The anion-exchangeresins differ by the following properties:

    ______________________________________                                        Amount of MBA     0.5 g    1 g      2 g                                       Ionic charges per ml of resin                                                                   36 μeq                                                                              114 μeq                                                                             218 μeq                                Sorption capacity per ml (BSA)                                                                  35 mg    91 mg    72 mg                                     ______________________________________                                    

Example 4

Preparation of an artion-exchange resin using MBMA as a cross linker.

1 g of N,N'-methylene-bis-methacrylamide (MBMA) is dissolved in 50 ml ofdimethylsulfoxide (DMSO). To this mixture 40 ml of an aqueous solutioncontaining 20 g of MAPTAC is added.

While stirring, 1 g of ammonium persulfate previously dissolved in 10 mlof distilled water is added. The obtained monomer solution is then usedto fill the silica pores (1 cm³ /g porous volume; 1200-1500 Å porediameter) and the resin is prepared according to the previous examplesexcept toluene is used as the non-aqueous solvent instead of paraffinoil.

The obtained anion-exchange resin shows the following characteristics:

Ion-exchange capacity: 201 μeq of quaternary amino groups per ml ofresin.

Sorption capacity for BSA: 112 mg/ml.

No non-specific adsorption of cationic proteins like cytochrome c arepresent.

Porosity factor for BSA (V_(e) /V_(t)): 0.53

Example 5

Preparation of anion-exchange resins with a different amount of MBMA.

Three different resins are prepared according to Example 4 withdiffering amounts of MBMA as a crosslinking agent.

When 100 ml of a DMSO-water solution is used, the amount of MBMA isvaried as follows: 0.5 g, 1 g and 2 g. Paraffin oil is used as thenon-aqueous (organic) solvent at 60° C.

The obtained resins show the following characteristics:

    ______________________________________                                        Amount MAPTAC  20 g      20 g      20 g                                       Amount MBMA    0.5 g     1 g       2 g                                        Ionic charges per ml                                                                         168 μeq                                                                              212 μeq                                                                              231 μeq                                 of resin                                                                      Sorption capacity per ml                                                                     114       106       76                                         Porosity factors                                                                             0.52      0.52      0.51                                       for BSA (V.sub.e /V.sub.t).                                                   ______________________________________                                    

It is demonstrated that the amount of crosslinking agent does not modifythe porosity of the three dimensional polymer at least within theexplored zone. The amount of ionic groups which depends on the amount ofthe main monomer remains also quite constant.

All of the above resins are stable to oxidizing agents, such ashypochlorites and peraceticacid.

Example 6

Preparation of strong cationic exchangers using silicas of differentporosity.

7 g of AMPS, 3 g of MAPTAC and 1 g of MBA are dissolved in 100 ml ofdistilled H₂ O. 1 g of ammonium persulfate is then added and thesolution is divided into two parts of 50 ml each. Separately, eachsolution is added to 50 g of dry silica having the following propertieslisted in the table below:

    ______________________________________                                        Particle                            Pore                                      Size         Surface Area                                                                             Porous Volume                                                                             Diameter                                  ______________________________________                                        Assay a                                                                              40-100 μm                                                                            25 m.sup.2 /g                                                                            1 cm.sup.3 /g                                                                           1250 Å                              Assay b                                                                              40-100 μm                                                                            10 m.sup.2 /g                                                                            1 cm.sup.3 /g                                                                           3000 Å                              ______________________________________                                    

All other operations are performed according to Example 1.

The following are the final properties of the cationic exchangers:

    ______________________________________                                                           Assay a Assay b                                            ______________________________________                                        Ionic charges per ml 92 μeq 89 μeq                                      Sorption capacity (cytochrome c)                                                                   86 mg     81 mg                                          Non-specific absorptions                                                                           negative  negative                                       ______________________________________                                    

This example demonstrates that the available porosity is independent ofthe silica quality. The choice of silica is more linked to itssensitivity to an alkaline media. For example, the alkaline sensitivityof silica having a surface area of 5 m² /g is 50% lower than when usinga sample having a surface area of 25 m² /g.

Example 7

Preparation of cation-exchangers using different amounts of anionicmonomer.

The aqueous solutions of monomers (100 ml) are composed of:

MAPTAC: 3 g (monomer to neutralize the silanol groups of silica)

AMPS: 7 g and 10 g (varying amounts of artionic monomer)

MBA: 1 g (crosslinker)

All other operations (mixing with silica, polymerization and recovery)are identical to those described on Example 1.

The final properties of the final cation-exchangers obtained are asfollows:

    ______________________________________                                        Quantity of AMPS     7 g       10 g                                           Ion-exchange groups per ml                                                                         92 eq     147 eq                                         Sorption capacity per ml                                                                           86 mg     120 mg                                         (cytochrome c)                                                                ______________________________________                                    

This example confirms that when the amount of functionalized monomer inthe initial solution is increased, the number of ion-exchange groups isproportionately higher. The sorption capacity for cytochrome c increasesas well.

Example 8

Preparation of a strong cation-exchange resin with MBMA as crosslinker.

0.5 g of MBMA are dissolved in 50 ml of DMSO while stirring. To thissolution 30 ml of aqueous solution containing 10 g of AMPS is added aswell as 6 ml of a 50% aqueous solution of MAPTAC.

The final volume is adjusted to 100 ml prior the addition of 1 g ofammonium persulfate at room temperature.

This solution of monomers is added dropwise to 100 g of dry poroussilica to fill completely the available porous volume (1 cm³ /g for apore size of 1250 Å). The remaining operations are identical to themethod described in Example 1.

The final cation-exchange resin shows the following characteristics:

    ______________________________________                                        ion-exchange groups per                                                                          123 μeq                                                 ml of resin                                                                   sorption capacity for                                                                            128 mg                                                     cytochrome c                                                                  porosity factor    0.82                                                       for lysozyme                                                                  resistance to oxidizing                                                                          Excellent even at a                                        agents (NaOCl)     concentrated form (1/10                                                       dilution of commercial)                                                       concentrated product.                                      ______________________________________                                    

Example 9

Preparation of a weak cation-exchange resin.

In 60 ml of distilled water, 6 ml of a 50% aqueous solution of MAPTAC, 1g of MBA and 10 ml of acrylic acid are dissolved.

The volume of the solution is then adjusted to 100 ml, the pH adjustedto about 4.5, and 1 g of ammonium persulfate is added at roomtemperature.

As described for other examples the solution of monomers is added to 100g of porous silica and then polymerized in a non-aqueouswater-immiscible solvent (e.g., paraffin oil, toluene, or methylenechloride).

The final characteristics of the resin are as follows:

    ______________________________________                                        Ion-exchange groups (carboxylates) per ml                                                                337 μeq                                         Sorption capacity for cytochrome c                                                                       118 mg                                             Non-specific adsorption    Excellent                                          ______________________________________                                         (chromatographic test)

Example 10

Preparation of non-ionic hydroxyl-containing resins for immobilizationof biologicals.

The monomers comprising the initial solution are the following:

    ______________________________________                                        Tris-hydroxymethyl-methyl-                                                                          Non ionic monomer                                       methacrylamide (THMMA)                                                        MAPTAC or DEAE methacrylamide                                                                       cationic monomer                                                              to neutralize the                                                             silanol groups.                                         MBA                   crosslinking agent                                      ______________________________________                                    

The composition of the solutions are:

    ______________________________________                                                     Assay a Assay b   Assay c                                        ______________________________________                                        THMMA          10 g      10 g      20 g                                       MAPTAC         1.5 g     --        2.5 g                                      DEAE-methacrylamide                                                                          --        2 g       --                                         MBA            2 g       3 g       2 g                                        ______________________________________                                    

All other operations (mixture with dry silica, polymerization andrecovery) are identical to those described in previous examples.

The final characteristics of the resins are:

Good passivation of the silica surface. No significant amount ofcationic proteins adsorbed in normal conditions of gel filtration.

V_(e) /V_(t) for bovine albumin is respectively 0.71, 0.74 and 0.61.

After chemical modification the resin is utilized to immobilize either adye (Cibacron Blue F3GA) or heparin.

Each affinity sorbents is very effective to purify human albumin andantithrombin III, respectively, in a single pass.

Example 11

Preparation of a cationic resin in the presence of polyethylene glycolas a pore inducer.

Two monomer solutions are prepared as described in Example 8. A solutionof 10 g of polyethylene glycol 6000 is added to one.

Final volumes are adjusted to 100 ml, pH adjusted to about 7 and then 1g of ammonium persulfate is added to both solutions.

The monomer mixture is added to porous silica (1200 Å pore diameter,40-100 μm particle diameter, 25 m² /g surface area), polymerization andrecovery are effected as described in previous examples. The obtainedresins show the following characteristics:

    ______________________________________                                                       +PEG-6000 (10%)                                                                           PEG-6000                                           ______________________________________                                        MAPTAC           20 g          20 g                                           MBMA              1 g           1 g                                           CATIONIC GROUPS  200           193                                            (μeq/ml)                                                                   SORPTION CAPACITY BSA                                                                          112           127                                            Ve/Vt β-lactoglobulin                                                                     0.578         0.511                                          Ve/Vt BSA        0.548         0.513                                          Ve/Vt Immunoglobulins G                                                                        0.495         0.481                                          ______________________________________                                    

This example demonstrates that, in spite of the same amount of initialmaterial (similar number of ionic groups), the porosity is influenced bythe presence of PEG-6000.

The exclusion limit is actually larger when PEG is added.

Example 12

Further separations of protein mixtures by ionic resins.

Two resins are used to show their ability to separate protein mixturesrapidly and efficiently:

a cationic resin (quaternary ammonium resin from Example 5).

an anionic sulfonated resin (see Example 8).

The cationic resin (201 μeq quaternary amino groups/ml) is packed in acolumn of 1 cm in diameter and 8 cm in length and then equilibrated witha 0.05M Tris-HCl buffer, pH 8.5. A sample containing 1 mg of cytochromec, hemoglobin, betalactoglobulin and ovalbumin is injected and separatedunder a salt gradient.

The results of the separation of the four components is given below(FIG. 1A). Separation is achieved under a flow rate of 120 ml/hour.

The artionic resin (138 μeq SO₃ groups/ml) is packed in a column of 1 cmin diameter and 7 cm in length and then equilibrated with a 0.05Macetate buffer, pH 4.5. A sample containing ovalbumin,betalactoglobulin, cytochrome c and lysozyme is injected and separatedunder a salt gradient.

The result of the separation of four components is given below (FIG.1B). Separation is achieved under a flow rate of 140 ml/hour.

Example 13

Demonstration of the need to neutralize the silanol group when preparinga cation-exchange resin.

Two aqueous solutions of monomer (100 ml each) are prepared according toExample 1 differing essentially by the presence of the cationic monomerMAPTAC.

Final composition of monomer solutions is as follows:

    ______________________________________                                                     Assay a    Assay b                                               ______________________________________                                        AMPS           10 g         10 g                                              MBMA           0.5 g        0.5 g                                             MAPTAC         3 g          0                                                 ______________________________________                                    

All the operations (mixing with silica, polymerization and recovery) areidentical to those described in the above-mentioned examples.

The final properties of the obtained cation-exchangers are as follows:

    ______________________________________                                                        Assay a   Assay b                                             ______________________________________                                        Ion-exchanger groups per ml                                                                     123 μeq  118 μeq                                      Sorption capacity per ml                                                                        128 mg      77 mg                                           (cyt.c)                                                                       Separation efficiency                                                                           excellent (see                                                                            no                                                                fig. below  separation                                      ______________________________________                                    

This result demonstrates the necessity to neutralize acidic silanolsthat disturb the separation mechanism.

Example 14

Influence of the amount of cationic monomer on the passivation of silicasurface.

To demonstrate that the amount of cationic monomer necessary toneutralize silanol groups (passivation is proportional to the surfacearea) a series of trials are effected with porous silicas with differentsurface area.

Silicas chosen are the following:

    ______________________________________                                                     Silica X 015                                                                             Silica X 075                                          ______________________________________                                        Surface area per g                                                                           25 m.sup.2   100 m.sup.2                                       Porous volume per g                                                                          1 cm.sup.3   1 cm.sup.3                                        Bead size      40-100 microns                                                                             40-100 microns                                    ______________________________________                                    

Trials are performed using different amounts of MAPTAC (cationicmonomer) copolymerized with a non-ionic acrylic monomer (THMMA).

After polymerization, the degree of passivation is estimated by themeasurement of non-specific adsorption of lysozyme.

                                      TABLE I                                     __________________________________________________________________________    COMPOSITION OF POLYMERS AND RELATED ANALYTICAL RESULTS                        Type of silica                                                                              X 075                                                                             X 075                                                                             X 075                                                                             X 075                                                                             X 075                                                                             X 015                                                                             X 015                                                                             X 015                               __________________________________________________________________________    Surface area/g                                                                              100 m.sup.2                                                                       100 m.sup.2                                                                       100 m.sup.2                                                                       100 m.sup.2                                                                       100 m.sup.2                                                                       25 m.sup.2                                                                        25 m.sup.2                                                                        23 m.sup.2                          Amount MAPTAC 0%  1.5%                                                                              3%  6%  12% 0%  1.5%                                                                              3%                                  Cross linking ratio                                                                         0%  10% 10% 10% 10% 0%  10% 10%                                 Non-specific ads. (Lysozyme)                                                                55 mg                                                                             13 mg                                                                             13 mg                                                                             0 mg                                                                              0 mg                                                                              15 mg                                                                             0 mg                                                                              0 mg                                Passivation ration level                                                                    -   ±                                                                              +   +   ++  -   +   ++                                  __________________________________________________________________________     ++ Indicates that the number of nonspecific absorptions is close to zero,     indicating an excellent passivation level.                                    + At a nonspecific adsorption of less than 10 mg, passivation is also         quite good.                                                                   ± Indicates that the passivation level is less than 15 mg, which in        most instances is not acceptable for use in chromatographic separation.       - Indicates that the passivation level is greater than 15 mg and thus the     material is not performing the separation function correctly and thus         cannot be used for chromatographic separation.                           

It is thus demonstrated that the level of non-specific adsorption forlysozyme (a strong cationic protein) is high when the MAPTAC is absent.The non-specific adsorption for silica with large surface ares (X 075,100 m² /g) is higher (55 mg/ml of resin) than the non-specificadsorption for silica X 015 (25 m² /g; 15 mg/ml of resin). A certainproportionality exists between the surface area and the original levelof non-specific absorptions. The amount of MAPTAC to decrease the levelof non-specific adsorption down to zero is also proportional to thesurface area available: 1.5% of MAPTAC is necessary with silica X 015(25 m² /g) whereas at least 6% is necessary to passivate silica X 075(100 m² /g).

Example 15

Preparation of an Anion Exchange Resin Based on Polystyrene.

10 g of methacrylamidopropyltrimethylammonium chloride, 2 g ofN-(1,1-dimethyl-2-phenyl)ethylacrylamide and 2 g ofN,N,-methylene-bis-methacrylamide are dissolved in 30 ml of dimethylsulfoxide. The volume of the solution is then increased to 50 ml byadding 20 ml of water. Under stirring, 0.3 g of2,2,-azobis-(2-amidinopropane)-hydrochloride is added at roomtemperature.

While shaking, the monomer solution is added dropwise to 50 g of porouspolystyrene (50-150 μm beads diameter, 300-400 Å pore diameter). Theexcess of monomer solution is thus eliminated by filtration undervacuum. The impregnated polystyrene beads are introduced into a closedcontainer and heated at 80°-90° C. for five hours to polymerize themonomer solution within the pores of the polystyrene matrix.

Finally the obtained material is washed extensively with ethanol toeliminate the excess monomers and, subsequently, with water.

The resulting resin showed the following characteristics:

Very hydrophilic material (in opposition to the totally hydrophobicnature and unwettability, of the polystyrene

Ion exchange capacity: 100 μeq/ml of resin

Sorption capacity for BSA: 70 mg/ml.

Example 16

Performance Characteristics of the Passivated Porous Support of thePresent Invention at High Flow Rates

The performance characteristics of the passivated porous support arecompared with those of other support materials under high solution flowrates (e.g., approaching 100 cm/h). In particular, the relative sorptioncapacity and productivity characteristics of DEAE-Spherodex™,DEAE-Trisacryl Plus™, DEAE-Trisacryl™, DEAE-Agarose-based sorbent, andpassivated porous supports of the present invention are illustrated inFIGS. 3A and 3B. The absolute sorption capacities at flow ratesapproaching 200 cm/h are compared for these supports in FIG. 4. The dataof FIG. 4 are generated for a 50 mM Tris buffer (pH 8.6) solution of BSA(5 mg/ml).

It can be seen from FIG. 3A, that the useful sorption capacity decreasesby half or more at flow rates between about 50 cm/h to about 100 cm/hfor Trisacryl, Trisacryl Plus and the Agarose-based sorbent. Bycontrast, the degree to which the useful sorption capacity of thepassivated porous supports of the present invention (e.g., thepassivated support of Example 2 or 4) is retained as flow rate increasescompares favorably with DEAE-Spherodex™ even at flow rates approaching100 cm/h (i.e., the useful sorption capacity remains substantiallyunchanged as a function of flow rate).

Moreover, the productivity, a measure of the amount of materialprocessed in the separation procedure per unit time, of the respectivesupports are compared in FIG. 3B. Again, the performance of thepassivated porous supports of the present invention compares favorablywith the DEAE-SPherodex™ sorbent. The passivated porous supports of thepresent invention are clearly superior to DEAE-Spherodex™, however, whentheir sorption capacities are compared on an absolute basis, as shown inFIG. 4.

Example 17

Preparation of an Anion-Exchange Resin Using a Surface-Protected (i.e.,Pre-coated) Silica Passivated Porous Support

Polystyrene pellets (10 g, average molecular weight about 400,000daltons) are dissolved in 100 ml of methylene chloride and then addeddropwise to 100 g of porous silica (40-100 μm diameter, 2000-3000 Å porediameter, 10 m² /g surface area and about 1 cm³ /g porous volume). Afterabout 30 minutes shaking the mixture is dried under an air stream atroom temperature until total evaporation of the chlorinated solvent(i.e., until a constant weight is observed). The obtained dry powder isthen heated at 190° C. overnight to permit the polystyrene to form ahomogeneous thin layer on the surfaces (internal and external) of thesilica.

Next, 20 g of methacrylamidopropyl trimethyl ammonium chloride (MAPTAC)and 1 g of N,N'-methylene-bis-methacrylamide are dissolved in 80 ml ofdistilled water and the pH of the solution is adjusted to 7.5.Separately, 1 g of ammonium persulfate is dissolved in 20 ml ofdistilled water. The two solutions are then mixed together at roomtemperature and added dropwise to 100 g of polystyrene-coated silica,obtained as described above. After shaking for about 30 minutes,paraffin oil (250 ml) is added to the mixture, along with 2 ml ofN,N,N',N'-tetramethylethylenediamine to polymerize the monomer solutioninside the silica pores. The resulting suspension is then heated at60°-70° C. to induce polymerization.

The passivated resin is then recovered by filtration. The oil iseliminated with an extensive washing with water containing 0.1-0.5% of anon-ionic detergent and then stored in a saline buffer at neutral pH.The product resin shows very similar ion-exchange characteristics asthose described in Example 2. Additionally, its sensitivity in strongalkaline media is much improved as measured by its weight loss after onenight of contact with 0.5M sodium hydroxide. The passivated resin ofthis example lost only about half as much weight as an artionic resinprepared from silica having an unprotected surface area.

Alternatively, the polystyrene can be coated on the surfaces of thematrix by polymerizing the vinyl monomer in situ, thus assuring that theinternal surfaces of even the smallest pores of the matrix are coatedwith protective polymer. The conditions for the polymerization of thevinyl monomer are well known to those of ordinary skill (e.g., see,Kirk-Othmer Concise Encyclopedia of Chemical Technology,Wiley-Interscience Publication, New York, pp. 1115-1117). After such anin situ polymerization, it is preferred that the coated support beheated overnight at 190° C., as described above, to provide ahomogeneous thin-film layer over the matrix.

In addition, the polystyrene may also contain substituents, particularlyat the 4-position of the phenyl ring, which can be non-ionic orionizable. For example, carboxylic acids, carboxylic acid esters oramides, sulfates, phosphates, N,N-dialkylcarboxamides, loweralkylamines, N,N-dialkylamines, quaternary ammonium groups, and the likecan be present on the polymer. Indeed, a 4-iodo substituent on all or aportion of the phenyl groups of polystyrene would allow a large host ofother functional group to be introduced by known methods (e.g.,formation of aryllithium, Grignard, or copper reagents followed byquenching with carbon dioxide or alkylation).

Moreover, passivation of the porous solid matrix having a thin-filmcoating of a synthetic organic polymer can also be achieved by othervariations in the procedure disclosed in the present invention, such asthe method of Example 15.

Example 18

Determination of Ion-Exchange and Protein Sorption Capacity ofPreparation of Anion-Exchange Resins Based on Passivated Porous SilicaSupport of Different Surface Areas

This example provides evidence that polymerization of the passivationmixture within porous silica matrices forms a three-dimensional polymernetwork or "lattice", as opposed to a thin, substantiallytwo-dimensional surface coating. Three anion-exchange sorbents wereprepared using the methods of the present invention, the differencesbetween the sorbents relating primarily to the pore sizes and henceinternal surface areas of the silica matrices. These silica substratecharacteristics are summarized in the following table:

    ______________________________________                                                     X-005    X-015   X-075                                           ______________________________________                                        Particle size (microns)                                                                      40-100     40-100  40-100                                      Porous volume (cm.sup.3 /g)                                                                    1          1      1                                          Pore size (Angstroms)                                                                        3000       1250    300                                         Surface area (m.sup.2 /g)                                                                     10         25     100                                         ______________________________________                                         Surface area is seen to increase as the pore size decreases, while porous     volume remains essentially constant.

The characteristics of the passivated ("Q-CPI") anion-exchange supportprepared from these silica base materials are summarized in thefollowing table:

    ______________________________________                                        Silica Matrix    X-005     X-015   X-075                                      ______________________________________                                        Particle size (microns)                                                                        40-100    40-100  40-100                                     Ionic groups (microeq/ml)                                                                      111       133     183                                        BSA capacity (mg/ml)                                                                           130       125      82                                        Sorption efficiency                                                                            1.17      0.94    0.45                                       ______________________________________                                    

The ion-exchange capacity (i.e., number of ionic groups) and BSAsorption capacity are seen to be relatively constant; in fact, thesevalues decrease somewhat as the surface area of the silica support isincreased). In particular, ion-exchange and BSA sorption capacities donot increase as the surface area of the silica increases (i.e., fromleft to right in the table). This supports the interpretation that thepolymeric lattice formed upon polymerization of the passivating solutionforms a three-dimensional, substantially pore-filling network, asopposed to a thin pore-wall surface coating.

Example 19

Preparation of an Anion-Exchange Resin Based on a Surface-Protected(i.e., Polystyrene-Precoated) Passivated Porous Silica Support

Polystyrene pellets (10 g, average molecular weight approximately 400kD) were dissolved in 10 ml of methylene chloride and then addeddropwise to 100 g of porous silica. The silica was characterized by aparticle diameter of 40 to 100 microns, a pore diameter of 2000 to 3000Angstroms, a surface area of 10 m² /g surface area, and a porous volumeof about 1 cm³ /g. After about 30 minutes of shaking, the mixture wasdried under an air stream at room temperature until total evaporation ofthe chlorinated solvent had occurred, as evidenced by the attainment ofa constant particle weight. The dry powder was then heated overnight at180° C. to permit the polystyrene to form a thin, homogeneous surfacelayer or coating on both the internal and external exposed surfaceregions of the silica. This polystyrene-coated silica so obtainedexhibited only a fraction of the sensitivity to alkaline media that wasexhibited by unprotected silica matrices. In particular, deposition ofthe protective polystyrene coat in this manner was observed to reducethe extent of silica leaching by a factor of at least 2 to 3.

Next, 0.5 g of N-1-methylundecyl-acrylamide (MUA) were dissolved in 100ml of pure ethanol, and the solution was added dropwise to 100 g of thepolystyrene-coated silica obtained as described above. After shaking forabout 30 minutes, the material was placed in a nitrogen stream underconditions that resulted in complete evaporation of the ethanol (again,as observed by attainment of constant solids weight).

Next, 1 g of N,N'-methylene-bis-methacrylamide was dissolved in 20 ml ofdimethylsulfoxide. To this solution, 20 g ofmethacrylamidopropyltrimethylammonium chloride (MAPTAC) were added, andthe total volume of the solution was adjusted to 80 ml by the additionof distilled water. Separately, 0.5 g of azo-bis-amidino-propane (asinitiator) was dissolved in 10 ml of distilled water and then added tothe solution of monomers. The volume of the latter was then adjusted to100 ml with water; 90 ml of this solution were then added dropwise tothe polystyrene-precoated silica.

This material (i.e., monomer-solution-impregnated polystyrene-precoatedsilica) was then placed under nitrogen and in a closed vessel at 80° C.for over two hours. The product so obtained was then washed extensivelywith water and water-compatible solvents to remove any unpolymerizedmaterial and other reaction byproducts.

The cationic (i.e., anion-exchange) resin so prepared exhibited afixed-charge density (i.e., ion-exchange capacity) of 150microequivalents/ml of quaternary amino groups. Its capacity forreversibly absorbing BSA was 125 mg/ml. Non-specific binding (expectedto be extensive and excessive for unpassivated, polystyrene-coatedsilica) was minimal for the material produced by the method of thepresent invention.

Example 20

Preparation of a Cationic Resin Based on a Porous Polystyrene Matrix

Porous polystyrene beads, characterized by a particle diameter of 50 to70 microns, a pore diameter of 1000 Angstroms, and a porous volume of1.6 cm³ /g, were obtained as a commercially available product fromPolymer Laboratories, Inc. (Amherst, Mass.). Five grams of these porouscrosslinked polystyrene beads were washed extensively with ethanol andthen dried under vacuum.

Separately, 61 mg of methylene-bis-methacrylamide were dissolved in 3.76ml of dimethyl sulfoxide. To this was added 2.44 ml of an aqueoussolution containing 1.3 g of methacrylamido-propyltrimethylammoniumchloride (MAPTAC) and 25 mg of azo-bis-amidino-propane. To thissolution, which was stirred gently under a nitrogen atmosphere at 4° C.,was added 1.5 ml of pure ethanol. This solution was then added dropwiseto the dry polystyrene beads until it was totally absorbed within theporous volume of the beads. After 30 minutes of shaking, the mixture wasstirred in a closed vessel under a nitrogen pressure at 85° C. for atleast 2 hours. After this period, the product beads were removed andwashed extensively with acidic, alkaline, and aqueous alcohol solutionsto remove reaction byproducts and uncopolymerized materials.

The anion-exchange resin product obtained in this manner was veryhydrophilic and contained cationic groups at a density of 124microequivalents/ml of settled resin volume. Protein sorption capacityas measured by uptake of bovine serum albumin (BSA) was between 30 and50 mg/ml of settled resin, depending on operating conditions.

Example 21

Preparation of a Passivated Cationic Resin Based on a Porous PolystyreneMatrix

Example 21 differs from the preceding Example 20 in its incorporation ofthe passivating monomer MUA into the mixture polymerized within thepores of the polystyrene support. As before, porous polystyrene beads,characterized by a particle diameter of 50 to 70 microns, a porediameter of 1000 Angstroms, and a porous volume of 1.6 cm³ /g, areobtained as a commercially available product from Polymer Laboratories,Inc. (Amherst, Mass.). Five grams of these porous crosslinkedpolystyrene beads are washed extensively with ethanol and dried undervacuum.

Separately, 61 mg of methylene-bis-methacrylamide are dissolved in 3.76ml of dimethyl sulfoxide. To this are added 2.44 ml of an aqueoussolution containing 1.3 g of methacrylamido-propyltrimethylammoniumchloride (MAPTAC) and 25 mg of azo-bis-amidino-propane. To thissolution, which is stirred gently under a nitrogen atmosphere at 4° C.,are added 1.5 ml of pure ethanol containing 50 mg ofN-1-methyl-undecylacrylamide (MUA) as a passivating ("neutralizing")monomer. This solution is then added dropwise to the dry polystyrenebeads until it is totally absorbed within the porous volume of thebeads. After 30 minutes of shaking, the mixture is stirred in a closedvessel under a nitrogen pressure at 85° C. for 2 hours or more. Afterthis period, the product beads are removed and washed extensively withacidic, alkaline, and aqueous alcohol solutions to remove reactionbyproducts and uncopolymerized materials.

The anion-exchange resin product obtained in this manner containscationic groups at a density of about 115 microequivalents/ml of settledresin volume. Protein sorption capacity as measured by uptake of bovineserum albumin (BSA) is about 80 mg/ml of settled resin. The resin isstable over a wide range of pH values (from 1 to 14) and can be usedadvantageously in the chromatographic separation of various proteinmixtures.

Example 22

Preparation of a Passivated Anionic Resin Based on a Porous PolystyreneMatrix

Example 22 differs from the preceding Example 21 in two respects: (i)its replacement (on a 1-for-1 basis by weight) of an anionic monomer(acrylamido-methyl-propane sulfonic acid sodium salt) for the cationicmonomer (MAPTAC) used in the passivating mixture polymerized within thepores of the porous polystyrene support, and (ii) its use ofN-(1,1,3,5-tetramethyloctyl)-acrylamide as opposed toN-1-methyl-undecyl-acrylamide (MUA) as the passivating or neutralizingmonomer.

As before, porous polystyrene beads, with a particle diameter of 50 to70 microns, a pore diameter of 1000 Angstroms, and a porous volume of1.6 cm³ /g, are obtained from Polymer Laboratories, Inc. Five grams ofthese porous crosslinked polystyrene beads are washed extensively withethanol and dried under vacuum.

Separately, 61 mg of methylene-bis-methacrylamide are dissolved in 3.76ml of dimethyl sulfoxide. To this are added 2.44 ml of an aqueoussolution containing 1.3 g of acrylamido-methyl-propane sulfonic acidsodium salt and 25 mg of azo-bis-amidino-propane. To this solution,which is stirred gently under a nitrogen atmosphere at 4° C., are added1.5 ml of pure ethanol containing 50 mg ofN-(1,1,3,5-tetramethyloctyl)-acrylamide as a passivating("neutralizing") monomer. This solution is then added dropwise to thedry polystyrene beads until it is totally absorbed within the porousvolume of the beads. After 30 minutes of shaking, the mixture is stirredin a closed vessel under a nitrogen pressure at 85° C. for 2 hours ormore. After this period, the product beads are removed and washedextensively with acidic, alkaline, and aqueous alcohol solutions toremove reaction byproducts and uncopolymerized materials.

The cation-exchange resin product obtained in this manner is veryhydrophilic and contains anionic (sulfonate) groups at a density ofabout 100 microequivalents/ml of settled resin volume. Protein sorptioncapacity as measured by uptake of lysozyme is about 95 mg/ml of settledresin. The anionic resin is stable over a wide range of pH values (from1 to 14) and can be used advantageously in the chromatographicseparation of various protein mixtures.

Example 23

Preparation of an Anion-Exchange Resin Using a Surface-Protected (i.e.,Pre-coated) and POE-Passivated Porous Silica Support

Polystyrene pellets (10 g, average molecular weight approximately 400kD) were dissolved in 10 ml of methylene chloride and then addeddropwise to 100 g of porous silica. The silica was characterized by aparticle diameter of 40 to 100 microns, a pore diameter of 2000 to 3000Angstroms, a surface area of 10 m² /g surface area, and a porous volumeof about 1 cm³ /g. After about 30 minutes of shaking, the mixture wasdried under an air stream at room temperature until total evaporation ofthe chlorinated solvent had occured, as evidenced by attainment of aconstant particle weight. The dry powder was then heated overnight at190°-200° C.

This polystyrene-coated silica was then suspended in 200 ml of anaqueous solution of 5% polyoxyethylene (POE) with an average molecularweight of about 600 kD. The mixture was stirred gently for about 5 hoursat 85° C. and then the excess solution was removed by filtration. Thesilica beads were then washed extensively with water to remove theexcess POE; the beads were finally rinsed twice with pure ethanol anddried.

Separately, 1 g of N,N'-methylene-bis-methacrylamide was dissolved in 20ml of dimethylsulfoxide under stirring. To this solution, 20 g ofmethacrylamidopropyltrimethylammonium chloride was added, and the totalvolume of the solution was adjusted to 80 ml by the addition ofdistilled water. Next, 0.5 g of azo-bis-amidino-propane was dissolved in10 ml of water and then added to the solution of monomers. The latterwas then adjusted to a total volume of 100 ml with water. Ninetymilliliters of this solution were then added dropwise to the precoatedPOE-treated dry silica. The silica, impregnated with monomer solution,was then placed in a closed vessel at 80° C. and the polymerization waseffected under nitrogen for two hours. The product so obtained waswashed extensively with water and water-compatible solvents at acidicand alkaline pH values to eliminate any unpolymerized materials andreaction by products.

The cationic (i.e., anion-exchange) resin so obtained exhibited anion-exchange capacity of 170 microequivalents/ml of quaternary ammoniumgroups and displayed a reversible BSA sorption capacity of 115 mg/ml. Nonon-specific binding was evident during a chromatographic separationconducted with the material.

Example 24

Preparation of an Anion-Exchange Resin Using a Surface-Protected (i.e.,Pre-coated) and PVP-Passivated Porous Silica Support

Polystyrene pellets (10 g, average molecular weight approximately 400kD) are dissolved in 10 ml of methylene chloride and then added dropwiseto 100 g of porous silica with characteristics described in the previousexample. After about 30 minutes of shaking, the mixture is dried underan air stream at room temperature until total evaporation of thechlorinated solvent has occured, as evidenced by attainment of aconstant particle weight. The dry powder is then heated overnight at190°-200° C.

This polystyrene-coated silica is then suspended in 200 ml of an aqueoussolution of 5% polyvinylpyrrolidone (PVP) with an average molecularweight of about 400 kD. The mixture is stirred gently for about 5 hoursat 85° C. and then the excess solution is removed by filtration. Thesilica beads are then washed extensively with water to remove the excessPOE; the beads are finally rinsed twice with pure ethanol and dried.

Separately, 1 g of N.N'-methylene-bis-methacrylamide are dissolved in 20ml of dimethylsulfoxide under stirring, To this solution, 20 g ofmethacrylamidopropyl-trimethylammonium chloride are added, and the totalvolume of the solution is adjusted to 80 ml by the addition of distilledwater. Next, 0.5 g of azo-bid-amidino-propane are dissolved in 10 ml ofwater and then added to the solution of monomers. The latter is thenadjusted to a total volume of 100 ml with water. Ninety milliliters ofthis solution are then added dropwise to the precoated POE-treated drysilica. The silica, impregnated with monomer solution, is then placed ina closed vessel at 80° C. and the polymerization is effected undernitrogen for two hours. The product so obtained is washed extensivelywith water and water-compatible solvents at acidic and alkaline pHvalues to eliminate any unpolymerized materials and reaction byproducts.

The cationic (i.e., anion-exchange) resin so obtained exhibits anion-exchange capacity of about 160 microequivalents/ml of quaternaryammonium groups and displays a reversible BSA sorption capacity of about120 mg/ml. Little or no non-specific binding is evident during achromatographic separation conducted with the material.

Example 25

Preparation of a Cation-Exchange Resin Using a Surface-Protected (i.e.,Pre-coated) and POE-Passivated Porous Silica Support

Polystyrene pellets (10 g, average molecular weight approximately 400kD) are dissolved in 10 ml of methylene chloride and then added dropwiseto 100 g of porous silica with the following characteristics: a particlediameter of 25 to 60 microns, a pore diameter of 3000 Angstroms, asurface area of 15 m² /g surface area, and a porous volume of about 1cm³ /g. . After about 30 minutes of shaking, the mixture is dried underan air stream at room temperature until total evaporation of thechlorinated solvent has occured, as evidenced by attainment of aconstant particle weight. The dry powder is then heated overnight at190°-200° C.

This polystyrene-coated silica is then suspended in 200 ml of an aqueoussolution of 5% polyoxyethylene and stirred gently for about 5 hours at85° C. The excess solution is removed by filtration. The silica beadsare then washed extensively with water to remove the excess POE; thebeads are finally rinsed twice with pure ethanol and dried.

Next, 1 g of N,N'-methylene-bis-methacrylamide, 1 g ofmethacrylamidopropyl-trimethylammonium chloride, and 18 g ofacrylamido-methyl-propane sulfonic acid sodium salt are dissolved in 90ml of a solvent mixture comprised of 20 ml of dimethylsulfoxide, 60 mlof water, and 10 ml of ethanol. To this solution 10 ml of watercontaining 0.5 g of azo-bis-amidinopropane are added. The final mixtureso obtained is then added dropwise to the "dry", polystyrene-protectedsilica. This silica, impregnated with monomer solution, is then placedin a closed vessel at 80° C. and the polymerization is effected undernitrogen for a period of at least 3 hours. The polyanionic product soobtained is then washed extensively as described in the immediatelypreceding examples.

The resin so obtained exhibits an ion-exchange capacity of about 100microequivalents/ml of sulfonate groups and displays a reversiblelysozyme sorption capacity of about 130 mg/ml.

It should be apparent to those skilled in the art that othercompositions and methods not specifically disclosed in the instantspecification are, nevertheless, contemplated thereby. Such othercompositions and methods are considered to be within the scope andspirit of the present invention. Hence, the invention should not belimited by the description of the specific embodiments disclosed hereinbut only by the following claims.

What is claimed is:
 1. A method of separating a preselected biologicalmolecule from a sample containing same comprising:(a) loading a column,packed with the passivated porous support comprising (i) a porousmineral oxide having interior and exterior surfaces substantiallycovered by a thin, protective polymer surface coating having innatehydrophobic groups that render said coating susceptible to undersirablenon-specific interaction with one or more biological molecules, (ii) apolymer network derived from a passivation mixture comprising a mainmonomer which is selected to provide a polymer network that has affinityfor a preselected biological molecule, a passivating monomer differentfrom said main monomer, and a crosslinking agent, said mixture havingbeen allowed to come into intimate contact with said surfaces of saidcoating such that on polymerization of said mixture said innate groupsof said coating become deactivated, resulting in the substantialelimination of said undersirable non-specific interaction, with a samplecontaining a preselected biological molecule to be separated from amixture; (b) passing an eluent solution through said loaded column toeffect the separation of said biological molecule.
 2. The method ofclaim 1 in which said biological molecule is a protein.
 3. Achromatographic method for the separation of biological moleculescomprising passing a sample containing a mixture of biological moleculesthrough a column containing the passivated porous support comprising (i)a porous mineral oxide having interior and exterior surfacessubstantially covered by a thin, protective polymer surface coatinghaving innate hydrophobic groups that render said coating susceptible toundesirable non-specific interaction with one or more biologicalmolecules, (ii) a polymer network derived from a passivation mixturecomprising a main monomer, a passivating monomer different from saidmain monomer, and a crosslinking agent, said mixture having been allowedto come into intimate contact with said surfaces of said coating suchthat on polymerization of said mixture said innate groups of saidcoating become deactivated, resulting in the substantial elimination ofsaid undersirable non-specific interaction.
 4. The chromatographicmethod of claim 3 wherein said column is selected from the groupconsisting of a packed column and a fluidized-bed column.